HIV reverse transcriptase (RT) has been an attractive target for HIV drug development, with 11 of the 20 approved drugs targeting RT DMA polymerase activity. However, HIV drug resistance is an increasingly serious clinical problem. New therapeutics are needed, especially those against unaddressed HIV targets, such as HIV RT-associated RNase H (RNH). RNH is underexplored for antiviral therapeutic discovery and development, and very few RNH inhibitors (RNHI) have been identified. This research program, HIV RNase H Natural Product Inhibitors, builds on a novel technology for plant cell culture as a source of novel antiviral agents. We have already identified several natural product RNHI with submicromolar antiviral activity. The proposed studies comprise three projects designed to develop and optimize these and other compounds to be identified from screening an existing 160,000 product library. The iterative research and development program combines the efforts of several investigators from academia and industry with considerable experience in HIV drug discovery and development. Project 1, Isolation and Optimization (Baroudy, Project Leader, Millenia Hope Inc) will isolate and purify natural products from plant cell cultures and carry out semisynthetic optimizations based on SAR developed in the other projects. Project 2, Biochemistry and Virology (Parniak, Program Director, University of Pittsburgh) will characterize the compounds for biological activity to generate data for use in SAR development, conduct detailed mechanism of action analysis, and screen the product library for new RNHI chemotypes. Project 3, Structural and Computational Biology (Arnold, Project Leader, CABM/Rutgers) will determine structures of RT complexed with RNHI for use along with data from Project 2 to develop an SAR to predict modifications to improve potency. Such modifications will be made in Project 1, then characterized in Projects 2 &3. This iterative process will continue until 2-3 lead candidates and 4-6 backups with low nM potency have been selected to enter extensive preclinical assessment. The research program will have significant impact on public health by developing new anti-HIV therapeutics for use in the treatment of patients infected with HIV strains resistant to the current clinically used drugs.